The manufacturing of recombinant protein-based biopharmaceuticals is a complex, labor and capital-intensive endeavor. Currently, mammalian cells are used for the production of most human proteins. Mammalian cells typically contain extensive post-translational modifications that may not be performed by unmodified prokaryotes and unmodified single-celled eukaryotes. Although mammalian cells such as Chinese hamster ovary cells and baby hamster kidney cells can faithfully biosynthesize most human proteins, the efficiency is dramatically lower than is achieved by bacterial or yeast cells.
Of the recombinant proteins currently marketed, fVIII is manufactured with the lowest efficiency and is by far the most expensive on a per unit mass basis (FIG. 1). Recombinant fVIII is the premiere treatment option for persons with the congenital X-linked bleeding disorder, hemophilia A. Treatment consists of 2-3 intravenous infusions per week of recombinant fVIII at a cost of approximately $100,000-$300,000 per year (Bohn R L, Avorn J, Glynn R J, Choodnovskiy I, Haschemeyer R, Aledort L M. Prophylactic use of factor VIII: an economic evaluation. Thromb Haemost. 1998; 79(5):932-7, incorporated herein in its entirety). Consequently, treatment access is limited to less than one-third of persons with hemophilia A worldwide. Historically, fVIII supply has been inadequate and price has remained exorbitant due to high research, development and manufacturing costs. One strategy for improving the care of hemophilia A, as well as other monogenic diseases that can be treated by protein replacement therapy, is to develop more efficient methods for recombinant protein manufacturing.
State-of-the-art recombinant h-fVIII products are produced typically by mammalian cells, e.g., BHK-21 or Chinese hamster ovary cells, in large-scale fermenting bioreactors. Several techniques may be used to maximize the production of recombinant h-fVIII including (1) amplification of the h-fVIII transgene using DHFR/methotrexate selection, (2) addition of fVIII stabilizing agents to the culture medium (e.g. bovine/human albumin or co-expression of vWf), and (3) maximizing cell growth/density by continuous-perfusion fermentation. FVIII may be purified from conditioned culture medium using a series of filtration, immunoaffinity, size-exclusion and ion-exchange chromatography steps. Often, viral inactivation procedures are incorporated into the purification protocol for added safety. Once purified, the bulk fVIII material may be formulated with stabilizing agents and may be freeze-dried prior to packaging. This standard manufacturing process is reviewed in Boedeker B G. Production processes of licensed recombinant factor VIII preparations. Semin Thromb Hemost. 2001; 27(4):385-94, incorporated herein by reference in its entirety.
First generation recombinant fVIII products were stabilized using human serum albumin that theoretically could harbor viral contaminants. To reduce the risk of viral contaminants, second and third generation fVIII products have emerged that are considered “animal-product free” and instead are stabilized with sucrose and other additives. Due to the perceived improved safety profile of newer generation recombinant products over both plasma-derived and first generation products, many previously-treated and the majority of previously-untreated patients have transitioned to second and third generation fVIII products. This demand has created multiple fVIII product shortages and lead to the implementation of strategies to temporarily ration fVIII supplies (Garber K. rFactor VIII deficit questioned. NatBiotechnol. 2000; 18(11):1133, incorporated herein by reference).
Several publications have stated that the standard level of recombinant human fVIII production is <1 unit/106 cells/day (Kaufman R J, Pipe S W, Tagliavacca L, Swaroop M, Moussalli M. Biosynthesis, assembly and secretion of coagulation factor VIII. Blood CoagulFibrinolysis. 1997; 8 Suppl 2:53-14.:S3-14, incorporated herein by reference). Typically, the final recombinant human fVIII product has a specific activity between 4,000-10,000 units per milligram protein and the cost of a single treatment for a 70 kg adult is $2,500-$5,000. Currently, fVIII products represent a 6-8 billion dollar annual market despite the fact that distribution is limited to less than one-third of the potential world market.